One critical mechanism of peripheral immunologic tolerance occurs through the process of T cell anergy, particularly in the tumor context. We previously have shown that upregulated expression of lipid kinases of the diacylglycerol kinase (DGK) family participate in the suppression of RasGRP-mediated Ras activation and contribute to T cell dysfunction in the anergic state. Other studies have implicated upregulation of additional negative regulatory signaling proteins, which may cooperate to maintain T cell dysfunction. Recently, through conditional knockout studies, gene expression profiling and ChIP-Seq experiments, we have identified the transcription factor EGR2 as a central regulator of expression of these negative regulatory factors in T cell anergy. Importantly, new EGR2-driven targets have emerged that are cell surface markers, which may allow flow cytometric identification of anergic cells. The major goal of this proposal is to determine the contribution o EGR2 towards peripheral tolerance to tumors. In Specific Aim 1 we will determine the effects of conditional deletion of EGR2 in peripheral T cells in vivo. Induction of T cell anergy, generation of anti-tumor immunity, and potential for evolving autoimmunity will be assessed. In Specific Aim 2, the relevance of a subset of EGR2- regulated genes in T cell biology will be investigated. We will focus on the secreted factor CCL1; the surface expression of CRTAM/LAG3/4-1BB; and the functional role of Semaphorin 7A. Whether combinations of these markers can identify anergic cells in the tumor microenvironment will be determined. In Specific Aim 3, expression of EGR2 and its targets in T cells infiltrating human melanoma will be evaluated, to move towards clinical relevance. Together, these studies will substantially broaden and deepen our understanding of EGR2 and T cell anergy in vivo, and are expected to uncover strategies that can have clinical application towards immunotherapy of cancer.